Useof anabolic agents anti-catabolic agents and antioxidant agents for protection treatment and repair of connective tissues in humans and animals

ABSTRACT

The present invention relates to compositions for the protection, treatment and repair of connective tissues in humans and animals comprising any or all of anabolic, anti-catabolic, and anti-oxidant agents, including aminosugars, S-adenosylmethionine, arachadonic acid, GAGs, pentosan, collagen type II, tetracyclines or tetracycline-like compounds, diacerin, super oxide dismutase, and L-ergothionine and to methods of treating humans and animals by administration of these novel compositions to humans and animals in need thereof.

FIELD OF THE INVENTION

[0001] The present invention relates to compositions for the protection,treatment and repair of connective tissues in humans and animals.

BACKGROUND OF THE INVENTION

[0002] The tissues of mammals, including humans, are in a constant stateof flux between the anabolic processes that build up tissues, and thecatabolic processes which degrade tissues. The state of health existswhen there is a balance between these two processes, and derangements ofthe balance produce disease. This holds true for all tissues of thebody. Connective tissues are of particular importance for severalreasons. First, they support the “functional cells” of the body, i.e.,epithelial, muscle and neural cells. Second, they play critical roles inintercellular communication, which is essential for multicellular life.

[0003] The inflammatory process occupies a key position in this balance.When injury to tissues occurs, inflammation initiates the biochemicalprocesses that result in tissue repair. Because inflammation results inthe symptoms of pain, inflammation, and swelling of the tissuesinvolved, it is often regarded by both patients and physicians as anabnormal and undesirable state, which should be treated and relieved assoon and as completely as possible. As a result, pharmacies are full of“anti-inflammatory drugs” (such as corticosteroids and the non-steroidalanti-inflammatory drugs, such as aspirin). Under certain circumstances,inflammation can indeed be destructive; however, it is important toremember that inflammation is closely linked with tissue healing.Indeed, inflammation is not easily categorized as strictly anabolic orcatabolic—it may have either effect. Its purpose in the body is toremove, dilute or wall-off the injurious agent(s). It also sets intomotion the biochemical processes that repair and reconstruct the damagedtissue. Because it is essential to healing, and because it can alsocause tissue destruction, inflammation and its mediators are importantfactors in the anabolic and catabolic balance.

[0004] One very important class of inflammatory mediators is theeicosanoid group. The eicosanoids are synthesized in the body fromessential fatty acids (“Fas”). Through a series of biochemicalreactions, the precursor fatty acids are modified to produceintermediate metabolites, arachadonic acid (“AA”), an omega-6 FA; andeicosapentanoic acid (“EPA”), an omega-3 FA. Eicosanoids produced fromarachidonic acid include the 2-series of prostaglandins and the 4-seriesof leukotrienes, which are generally proinflammatory. The eicosanoidsderived from EPA, such as the 3 series prostaglandins andhydroxyeicosapentaenoic acid (“HEPE”), are less inflammatory than thosederived from AA. In addition, such eicosanoids may even haveanti-inflammatory effects.

[0005] As a class, the eicosanoids are short-lived and locally active.They are responsible for the initial events of inflammation, includingvasodilation, increased vascular permeability, and chemotaxis. Moreover,the eicosanoids are instrumental in the early steps of the healingprocess. For example, the eicosanoids trigger the release of cytokinessuch as TGF-β, which in turn stimulates the migration and proliferationof connective tissue cells, and the deposition of extracellular matrix.Specific constitutive eicosanoids also have protective effects in thegastrointestinal mucosa and kidney, because they maintainglycosaminoglycan synthesis and normal perfusion of these organs.

[0006] Because of anabolic processes such as these, and because of theinfluence of natural anti-catabolic and anti-oxidant agents in the body,the outcome of the majority of cases of inflammation is resolution ofthe injury and healing of the damaged tissues. Only in pathologicsituations does inflammation itself become a contributor to disease.

[0007] Research on the therapeutic use of eicosanoid precursor FAs(including cis-linoleic and alpha-linolenic acids, the so-called omega-3and omega-6 fatty acids) has been primarily directed towards their useas competitive inhibitors of the synthesis of eicosanoids, andtherefore, their anti-inflammatory effects. Except in cases of severe orabsolute dietary deficiency, little attention has been given to thebeneficial, anabolic effects that the eicosanoids have in connectivetissues. However, naturally occurring “subclinical” deficiencies ofeicosanoids probably contribute significantly to disease, and are underdiagnosed. For example, the enzyme delta-6-desaturase is responsible forthe committed step in the synthesis of AA. Activity of this enzyme,(delta-6-desaturase) decreases with age. This is likely to prove asignificant factor in the increased incidence of connective tissuedysfunction in older population segments since a deficiency of AA woulddecrease anabolic processes and allow catabolic events to dominate.

[0008] Given the importance of inflammation in the healing of tissues,and the protective role that some eicosanoids play, it is not surprisingthat pharmaceuticals that decrease inflammation by blocking eicosanoidproduction should also have negative effects on healing and anabolicprocesses. It has long been known that corticosteroid drugs, which arestrongly anti-inflammatory, also delay healing and decrease theproduction of extracellular matrix components. This is because cortisoland related compounds stabilize cell membranes and therefore inhibit therelease of phospholipase A2, the precursor of AA. Recently attention hasturned to the non-steroidal anti-inflammatory drugs (“NSAIDs”). Numerousstudies have shown that NSAIDs, like corticosteroids, can decrease thesynthesis of matrix components by connective tissue cells, because theyinhibit prostaglandin endoperoxide synthase, and thus block thecyclooxygenase pathway.

[0009] Since the inflammatory process is the sine qua non of tissuehealing, and since the eicosanoids are the mediators of the inflammatoryprocess, the use of AA (and other eicosanoid compounds) is a novelapproach to therapy of injured tissues. Kirkpatrick et al. investigatedthe use of prostanoid precursors on chick embryonic cartilage in organculture and found no significant effects. [Kirkpatrick, C. J., “Effectsof Prostanoid Precursors and Indomethacin on Chick Embryonic CartilageGrowth in Organ Culture,” Expl. Cell Biol., 51:192-200 (1993)]. Theexperimental model in this work may have contributed to the absence ofsignificant effects, because avian cartilage and embryonic cartilagediffer significantly from mammalian, postnatal cartilage. For example,embryonic cartilage of any species is hypermetabolic and anabolic tobegin with because it is in a period of exponential growth. Kent et al.examined the effects of AA in lapine cartilage and found a positiveeffect, although previous and subsequent research failed to confirmthis. [Kent, L. et al., “Differential Response of Articular ChondrocytePopulations to Thromboxane B2 and Analogs of Prostaglandin CyclicEndoperoxidases,” Prostaglandins, 19:391-406 (1980)]. Kirkpatrick andGardner found that AA and various metabolites of AA had insignificant orinhibitory effects on biosynthesis. [Kirkpatrick C. J. and Gardner, D.L., “Influence of PGA1 on Cartilage Growth,” Experientia, 33(4):504(1976)]. These variable results are not unexpected, since the balancebetween anabolic and catabolic processes in the body is delicate andeasily perturbed. Phan et al., suggest that products of AA via thecyclooxygenase pathway are anti-fibrogenic while AA products via thelipoxygenase pathway are pro-fibrogenic. This phenomenon demonstratesthe complexity of the eicosanoids' interactions.

[0010] Catabolic events are typically mediated in the body by enzymesthat break apart body constituents. Catabolism is essential for healthand deficiency of necessary enzymes results in disease, such as theso-called storage diseases like mucopolysaccharhidosis. Excessivecatabolism may also result in the breakdown of tissues and lead todisease, as in degenerative diseases like osteoarthritis or autoimmunediseases like multiple sclerosis. Various anti-catabolic substances inthe body help contain and balance catabolism. For example, chondroitinsulfate counteracts metalloproteinases that catabolize collagen andproteoglycans in the cartilage matrix. Similarly, alpha-one anti-trypsininhibits the effects of elastase, which contributes to alveolarbreakdown in emphysema.

[0011] Oxidative damage also has an impact on the balance of anabolismand catabolism in the body. This damage is the result of the effects offree radicals, substances that have an unpaired electron. Free radicalsform constantly in the body as the result of normal reactions like theproduction of ATP. They also form during the inflammatory process. Freeradicals cause cellular damage because they are highly chemicallyreactive. Because they have only a single electron, (a condition thatnature abhors as it does a vacuum), these substances “steal” electronsfrom molecules in their vicinity. The molecules making up cellstructures, such as the cell membrane or DNA are thereby renderedelectron-deficient. The deficiency of electrons in turn makes the cellstructure unstable and cell dysfunction occurs, including manufacture ofabnormal proteins, cell rupture, and cell death. Oxidative damage isimplicated in many catabolic events in the body, including the agingprocess. Anti-oxidants, such as vitamin C, vitamin E, superoxidedismutase (SOD), selenium, and glutathione are substances that scavengefree radicals before oxidative damage occurs. In the sense that theyprevent cell damage, anti-oxidants are a specific type of anti-catabolicagent.

[0012] The body also contains anabolic compounds that stimulate tissuegrowth. Glucosamine is an amino sugar naturally formed in the body fromglucose. When supplied exogenously, glucosamine stimulates connectivetissue cell synthesis, and thereby increases the amounts of normalextracelluiar matrix. Glucosamine is also the building block forglycosaminoglycans in cartilage and other connective tissues. Supplyingadditional glucosamine thus supplies the body with extra raw materialsfor matrix synthesis in connective tissues. Other examples of anaboliccompounds in the body include somatotropin, which stimulates proteinsynthesis, and the somatomedins or insulin-like growth factors, whichstimulate the proliferation of chondrocytes and fibroblasts and enhancematrix synthesis.

[0013] The actions and interactions of these compounds are complex. Agiven compound may have different effects in different tissues. Forexample, somatotropin increases protein synthesis (anabolism), but alsospeeds fat breakdown (catabolism). The effects that a particularcompound or combination of compounds will have depend on many factors,including route of administration, dosage, and duration of therapy.

[0014] Previous researchers have investigated the use of individualcompounds for their anabolic, anti-oxidant or anti-catabolic effects.Glucosamine has been found in cell culture to stimulate connectivetissue cells to produce the components of the matrix: collagen andglycosaminoglycans (GAGs). [Jimenez, S., “The Effects of Glucosaminesulfate on Chondrocyte Gene Expression,” Eular Symposium, Madrid October1996 Proceedings, page 8-10]. S-adenosylmethionine is known toparticipate in several synthesis reactions, including the sulfation ofGAGs. [Champe, P. Biochemistry, 2^(nd) edition, J. B. Lippincott Co,Philadelphia, 1994, pp. 248, 250, 265]. Arachadonic acid has been foundto stimulate corneal healing. [Nakamura, M., “Arachidonic AcidStimulates Corneal Epithelial Migration”, J. Ocul. Pharmacol.,Summer:10(2): 453-9 (1994)]. These compounds therefore have anaboliceffects.

[0015] Chondroitin sulfate has been shown to inhibit degradativeenzymes, including the metalloproteinases that destroy cartilage matrix.[Bartolucci, C., “Chondroprotective action of chondroitin sulfate,” Int.J. Tiss. Reac., XIII(6):311-317 (1991)]. Studies with pentosan sulfatehave shown that it prevents complement-mediated damage in a rabbitmyocardial cells. [Kilgore, K., “The Semisynthetic PolysaccharidePentosan Polysulfate Prevents Complement-Mediated Myocardial Injury inthe Rabbit Perfused Heart,” J. Pharmocol. Exp. Ther., 285(3):987-94(1998)]. Oral administration of collagen type II has been shown todecrease the deleterious immune response that destroys joint tissue inrheumatoid arthritis. Tetracycline analogues are potent inhibitors ofmatrix metalloproteinases. [Ryan, M., “Potential of Tetracyclines toModify Cartilage Breakdown in Osteoarthritis.” [Curr. Opin. Rheumatol.,8(3): 238-47 (1996)]. Diacerein modifies the inflammatory process byinhibiting interleukin-1 activity, and also by direct effects onlymphocytes and neutrophils. [Beccerica, E., “Diacetylrhein and rhein:in vivo and in vitro effect on lymphocyte membrane fluidity,” Pharmocol.Res., 22(3):277-85 (1990); Mian, M., “Experimental Studies onDiacerhein: Effects on the Phagocytosis of Neutrophil Cells fromSubcutaneous Carregeenan-Induced Exudate,” Drugs Exp. Clin. Res.,13(11):695-8 (1987); Spencer, C., “Diacerein”, Drugs, 53(1):98-106(1997)]. These compounds can be classed as anti-catabolic agents.

[0016] L-ergothionine scavenges hydroxyl radicals and may inhibitsinglet oxygen formation, [Han JS. “Effects of Various ChemicalCompounds on Spontaneous and Hydrogen Peroxide Induced Reversion inStrain TA104 of Salmonella typhimuriu.” Mutant Res., 266(2):77-84(1992)], while superoxide dismutase scavenges superoxide radicals[Mathews C., Biochemistry 2^(nd) ed., Benjamin/Cummings Pub. Co., MenloPark Calif., 1996, page 551]. These compounds can be classified asanti-oxidants.

[0017] Although these compounds have been investigated individually, toour knowledge no one other than the present inventors has examined theeffects of certain combinations of any or all of anabolic,anti-catabolic and anti-oxidant agents to maintain health and to promotehealing. According to the present invention, combinations of theseagents can be used to maximize appropriate anabolic effects (healing)and decrease undesirable catabolic effects (degradation) and oxidativedamage, while at the same time, causing minimal or no adverse reactions.Therefore, it can be seen that there exists a need to providecompositions that will make use of the beneficial effects ofcombinations of anabolic agents, anti-catabolic agents and anti-oxidantagents for the maintenance and repair of connective tissues in humansand animals.

SUMMARY OF THE INVENTION

[0018] The present invention provides novel compositions and methods oftreating repairing, and preventing damage to connective tissues inhumans and animals using such compositions. Therefore, it is an objectof the invention to provide novel compositions of any or all ofanabolic, anti-catabolic, and anti-oxidant agents for the protection,treatment and repair of connective tissues in humans and animals.

[0019] It is another object of the present invention to provide methodsof treating and repairing connective tissue in humans and animals withcompositions containing any or all of anabolic, anti-catabolic, andanti-oxidant agents.

[0020] It is still another object of the present invention to providecompositions any or all of anabolic, anti-catabolic, and anti-oxidantagents selected from the group consisting of aminosugar,S-adenosylmethionine (SAMe), arachadonic acid (AA), GAG, pentosansulfate, collagen type II, tetracyclines, diacerin, super oxidedismutase (SOD), and L-ergothionine.

[0021] It is a further object of the present invention to providecompositions to repair, treat, and prevent damage to connective tissuein humans and animals that contain one or more of the elements selectedfrom the group consisting of aminosugar, SAMe, arachidonic acid, GAG,pentosan sulfate, collagen type II, tetracyclines, diacerin, SOD, andL-ergothionine.

[0022] These and other objects of the present invention are apparentfrom the detailed descripton and claims below.

[0023] Priority Claim:

[0024] In connection with this application, priority is claimed to thefollowing provisional application: THE USE OF ANABOLIC AGENTS,ANTI-CATABOLIC AGENTS AND ANTIOXIDANT AGENTS FOR PROTECTION, TREATMENTAND REPAIR OF CONNECTIVE TISSUES IN HUMANS AND ANIMALS, U.S. Ser. No.60/074,594, filed Feb. 13, 1998.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 provides a detailed description of the biosynthetic pathwayfor the creation of GAGs such as chondroitin sulfate.

[0026]FIG. 2 is the molecular structure of SAMe and its immediateprecursor.

[0027]FIG. 3 provides a simplified diagram of the function of SOD.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The compositions of the present invention, used to treat, repair,and prevent damage to connective tissue, consist of anabolic,anti-catabolic, and anti-oxidant agents selected from the groupconsisting of glucosamine, SAMe, AA, chondroitin sulfate, pentosansulfate, collagen type II, tetracyclines, diacerin, SOD, andL-ergothionine. In addition, the present invention covers methods ofadministering these novel compositions to humans and animals in needthereof.

[0029] Glucosamine—an example of an aminosugar—is naturally formed inthe body from glucose. When supplied exogenously, glucosamine stimulatesconnective tissue cell synthesis, increasing the amounts of normalextracellular matrix. Glucosamine is also the building block forglycosaminoglycans (“GAGs”) in cartilage and other connective tissues,thus, supplying additional glucosamine supplies the body with extra rawmaterials for matrix synthesis in connective tissues. The aminosugarcomponent of the compositions of the present invention may comprisenatural, synthetic or semi-synthetic aminosugars including but notlimited to salts of glucosamine including glucosamine hydrochloride andglucosamine sulfate, and N-acetylglucosamine and salts and/or mixturesthereof. In addition, the term aminosugar is also used herein toencompass aminosugars that may have been chemically modified yet retaintheir function. Such chemical modifications include but are not limitedto esterification, sulfation, polysulfation, acetylation, andmethylation. Moreover, it is contemplated that the term aminosugar canextend to any composition of matter that is insubstantially differentfrom the aminosugar as above-described.

[0030] The GAG component of the compositions of the present inventionmay comprise natural, synthetic or semisynthetic GAGs, GAG-likecompounds, or GAG precursors, including but not limited to chondroitin,hyaluronic acid, glucuronic acid, iduronic acid, keratan sulfate,heparan sulfate, dermatin sulfate, and fragments, salts, and mixturesthereof. In addition, the term GAG as used herein further encompassesGAGs that have been chemically altered yet retain their function. Suchmodifications include but are not limited to esterification, sulfation,polysulfation, and methylation. In fact, sulfated GAGs are a preferredcomponent of the compositions of the present invention. Hence,mono-sulfated and polysulfated (or oversulfated) GAGs are preferred GAGcomponents of the compositions of the present invention. The term GAGsalso is intended to encompass alternative nomenclature for the samegroup of above-described compounds—e.g., mucopolysaccharides,proteoglycans, and heparanoids. In addition, the GAG or GAG-likecomponent of the compositions of the present invention may be derivedfrom plant or animal sources, including but not limited to beechwoodtree, to forms of animal cartilage including shark cartilage, bovinetrachea, whale septum, and porcine nostrils, and to invertebrates suchas Perna canaliculus and sea cucumber.

[0031] Chondroitin sulfate is a preferred GAG. Chondroitin sulfate isthe most abundant glycosaminoglycan in articular cartilage and is alsopresent in many other connective tissues in the body. Additionally,chondroitin sulfate competitively inhibits degradative enzymes thatdegrade connective tissues under conditions of abnormal, excessiveinflammation. Chondroitin sulfate is a polymer composed of repeatingunits of glucuronic acid and sulfated galactosamine. [Lester M.Morrison, M.D. and O. Arne Schjeide, Ph.D., Coronary Heart Disease andthe Mucopolysaccharides (Glycosaminoglycans) 12 (1974); Philip C. Champeand Richard A. Harvey, Lippincott's Illustrated Reviews: Biochemistry,148-50 (2^(nd) ed. 1994)]. One of ordinary skill in the art understandsthat chondroitin sulfate must have at least two, and potentially many,of these repeating units of glucuronic acid and sulfated galactosamine.

[0032]FIG. 1 provides a detailed description of the biosynthetic pathwayfor the creation of GAGs, such as chondroitin sulfate. In addition, thepresent invention may include fragments of GAGs, such as fragments ofchondroitin sulfate. One of ordinary skill in the art at the time theinvention understands that “fragments of glycosaminoglycans” are groupsof saccharides that constitute less than two repeating units of theglycosaminoglycan. Hence, it is understood that fragments of thesesubstances would be composed of groups of saccharides that constitutefewer than two of the repeating units of the respective polymer. Forexample, one of ordinary skill in the art understands that fragments ofchondroitin sulfate are molecules composed of the saccharides thatcomprise the repeating units of chondroitin sulfate, but that arepresent in groups of less than the two repeating units described above.Thus, a molecule composed of a glucuronic acid and sulfatedgalactosamine would constitute a fragment of chondroitin sulfate.Indeed, there are eight different disaccharide structures that mayconstitute fragments of chondroitin sulfate. [Timothy E. Hardingham andAmanda J. Fosang, Proteoglycans: Many Forms and Many Functions, FASEBJ., 6:861-862 (1992)].

[0033] Other naturally occurring glycosaminoglycans may be used in thisinvention, for example, hyaluronic acid. Also, fragments of theglycosaminoglycans may also be utilized. A person of ordinary skill inthe art understands the terms “fragments of chondroitin,” “fragments ofchondroitin sulfate,” “fragments of chondroitin salts,” “fragments ofglycosaminoglycan” and “chondroitin sulfate fragments,” and furtherunderstands them to mean groups of saccharides (or salts thereof) thatconstitute less than two repeating units of the glycosaminoglycan.

[0034] One of skill would expect that fragments of chondroitin sulfate,for example, would have the same utility as chondroitin sulfate itself.Chondroitin sulfate is broken down into smaller units within the body,and that it is reformulated in the production of cartilage and otherconnective tissue. Therefore, it is understood that the body utilizesfragments of chondroitin sulfate in the same manner as it utilizeschondroitin sulfate itself. The same is true with respect to “fragmentsof chondroitin,” “fragments of chondroitin salts,” and “fragments ofglycosaminoglycan.” Each of chondroitin, chondroitin salts and otherglycosaminoglycans, if ingested, is broken down by the body andreformulated in the production of cartilage and other connective tissue.Therefore, the body utilizes fragments of chondroitin in the same manneras it utilizes chondroitin itself, utilizes fragments of chondroitinsalts in the same manner as it utilizes chondroitin salts, and utilizesfragments of glycosaminoglycans in the same manner as it utilizesglycosaminoglycans.

[0035] Moreover, it is intended that the term GAG can extend to anycomposition of matter that is insubstantially different from the GAGs asabove-described. An example of such a GAG-like compound that is withinthe scope of the present invention is pentosan polysulfate (PPS) as wellas salts thereof such as calcium-derived PPS and sodium PPS.Accordingly, a preferred GAG-like compound that may be used in thecompositions of the present invention is PPS.

[0036] PPS is a semi-synthetic polysulfated xylan that is a sulfatedform of a compound extracted from beechwood hemicellulose consisting ofrepeating units of (1-4) linked β-D-xylano-pyranoses. More specifically,PPS is produced by extracting these hemicellulose compounds via a seriesof chemical reactions from the wood, and then adding numerous sulfategroups to the purified polysaccharide chains. This process results inlow molecular weight linear polysaccharide chains that carry numerousnegatively charged sulfate groups. PPS is a semi-synthetic heparinoidthat is considered an oversulfated form of a GAG.

[0037] There are several forms of PPS that display the above-describedactivities. Sodium PPS and a calcium-derived PPS (called CAPPS) may bothbe used to accomplish the functions of PPS. Each of these forms of PPSexhibit GAG-like activity, and will hereinafter be referred to asGAG-like compounds.

[0038] Pentosan's mechanism of action can be summarized as follows:

[0039] 1. Anti-inflammatory activities through stabilization andimprovement of micro-circulation in the inflamed tissues and throughanti-Complement effects (decreases the release of the humoral mediatorsof inflammation called the Complement cascade).

[0040] 2. Inhibition of chemotaxis of granulocytes, which are whiteblood cells that contribute to inflammation.

[0041] 3. Stimulatory effect on proteoglycan synthesis.

[0042] 4. Stimulatory effects on hyaluronic acid synthesis by synovialfibroblasts.

[0043] 5. Potent inhibition of catabolic enzymes including, humangranulocyte elastase (noncompetitive inhibition), hyaluronidase(competitive inhibition), chondroitin-4-sulfatase andN-acetyl-glucosaminidase at concentrations much more lower than that ofNSAIDs.

[0044] Other synthetic or semi-synthetic glycosaminoglycans orglycosaminoglycan-like compounds, such as polysulfatedglycosaminoglycans, may be used in this invention.

[0045] Diacerein, a recently recognized organic compound found in plantsof the genus Cassia has anti-inflammatory effects through inhibition ofinterleukin-1β; consequently collagenase production in articularcartilage is reduced. It reduces the fibrinolytic activity of synovialfibroblasts as well. It also dose-dependently inhibits chemotaxis(attraction of white blood cells) and superoxide anion production (thisis one of the “toxic oxygen species” or “free radicals”). These harmfulcompounds occur spontaneously in the body, especially during destructiveinflammation. Diacerein has analgesic and antipyretic activities. Itreduces the turnover of chondroitin-4-sulfate resulting in a decrease inthe ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. (This ratiois pathologically increased in arthritic joints.) It mildly increasesprostaglandin synthesis, which allows it to have protective effects onthe gastric mucosa.

[0046] S-adenosylmethionine (SAMe) is an important endogenous compound,resent throughout the body, and taking part in a great number ofbiologic reactions such s transsulfation reactions. In this role it isan important reactant in the synthesis of many structural components ofconnective tissues, including proteins and proteoglycans. Thus, SAMe hassignificant anabolic effects which would enhance the actions of otheranabolic agents. SAMe also has anti-inflammatory effects by virtue ofits antioxidant action.

[0047] SAMe is compound synthesized in the body from adenosinetriphosphate (“ATP”) and methionine (FIG. 2). It is present in manytissues, including the central nervous system. The primary CNS functionof SAMe is to donate methyl groups in the reactions synthesizing variouscrucial compounds, including neurotransmitters and phospholipids. Forexample, SAMe facilitates the conversion of phosphatidylethanolamine tophosphatidylcholine, which forms part of the inner, lipid layer of theplasma membrane. In so doing, SAMe increases membrane fluidity andenhances effectiveness of receptor/ligand binding. [Champ and Harvey,Biochemistry, 1994; Stramentinoli, G., “Pharmacologic Aspects ofS-Adenosylmethionine,” American J. Med., 83(5A):35 (1987); Baldessarini,F., “Neuropharmacology of S-Adenosyl Methionine,” American J. Med.,83(5A):95 (1987); Carney, M., “Neuropharmacology of S-AdenosylMethionine,” Clin. Neuropharmacol., 9(3):235 (1986); Janicak, P.,“S-Adenosylmethionine in Depression,” Alabama J. Med. Sci. 25(3):306(1988)]. These functions may also pertain to other methyl donors such asbetaine (trimethylglycine), 5-methyltetrahydrofolate, folic acid, anddimethylglycine. [Champ and Harvey, Biochemistry, 1994].

[0048] Superoxide dismutase is an enzyme present naturally in thetissues of animals, which has recently been investigated as an agent inthe management of inflammation. It acts by intercepting toxic oxygenradicals in the intracellular space during destructive inflammatoryprocesses. It does not inhibit prostaglandin biosynthesis, but stops theoverproduction of prostaglandins resulting from destructiveinflammation. Some of its effects include inhibition of edema formationand inhibition of acute signs of inflammation and the secondaryarticular changes (stiffness and calcification) in adjuvant-inducedarthritis. Having no analgesic effects, it does not contribute to theoveruse of the affected joints that eventually leads to more damage ofthe articular cartilage, as NSAIDs can. Also, it has no adverse effectson the cardiovascular, central nervous or endocrine systems. FIG. 3provides a simplified diagram of the function of SOD.

[0049] L-ergothionine is an intracellular antioxidant naturallyoccurring in plants and animals, but not synthesized in human bodies: itcomes only from dietary sources. The antioxidant properties ofL-ergothionein appear to be related to its ability to scavenge reactiveoxygen species (free radicals), chelate various metallic cations,activate antioxidant enzymes such as glutathione peroxidase (SeGPx) andmanganese superoxide dismutase (Mn SOD) and to inhibitsuperoxide-generating enzymes such as NADPH-Cytochrome C reductase, andto affect the oxidation of various hemoproteins such as hemoglobin andmyoglobin. Because all body tissues depend on these two oxygen carriermolecules, this characteristic is extremely beneficial. [Brummel, M. C.,“In Search of a Physiological Function for L-ergothioneine,” Med.Hypotheses, 18(4):351-70 (December 1985); Brummel, M. C., “In Search ofa Physiological Function for L-ergothioneine,—II,” Med. Hypotheses,30(1):39-48 (Sept. 1989); Han, J. S., “Effects of Various ChemicalCompounds on Spontaneous and Hydrogen Peroxide-Induced Reversion inStrain TA104 of Salmonella typhimurium,” Mutat. Res., 266(2):77-84(April 1992); Arduini, A., “Possible Mechanism of Inhibition ofNitrite-Induced Oxidation of Oxyhemoglobin by Ergothioneine and UricAcid,” Arch. Biochem. Biophys., 294(2):398-402 (May 1992)].

[0050] Collagen Type II also has beneficial effects that help maintainthe normal balance between anabolism and catabolism. Specifically,connective tissue diseases may result from autoimmune processes, inwhich the immune system attacks and catabolizes the individual's ownconnective tissues as if it were a “foreign invader.” Oraladministration of collagen Type II can desensitize the immune system,preventing further attack and normalizing immune responses in theseindividuals. This decreases catabolic processes in the connectivetissues and maximize anabolism. Ingestion of collagen type II presentsthis molecule to the immune cells in the gut-associated lymphoid tissues(GALT, a.k.a., Peyer's patches). Interactions between the collagenmolecule and specific cells within the GALT activates mobile immunecells called T suppressor cells. These cells, in turn, moderate thedestructive immune reaction against the individual's own collagen typeII (in connective tissues).

[0051] Compounds in the tetracycline family include tetracycline,doxycycline, tetracycline analogs, and “tetracycline-like” compounds,and have been used therapeutically for their anti-microbial effects.Current research has focused on “tetracycline-like” compounds whichpossess insignificant antimicrobial effects, but with anti-cataboliceffects. Specifically, “tetracycline-like” compounds are polycycliccompounds that inhibit tissue metalloproteinases which degradeextracellular matrix components including collagen and proteoglycans yethave insubstantial anti-microbial effects. This function of thesecompounds, as well as other compounds in the tetracycline family, may berelated to the ability of these compounds to chelate calcium and zincions. For example, doxycycline has been shown to inhibit collagenaseactivity in articular cartilage.

[0052] Although the effects of these compounds have been investigated inisolation, the present invention comprises novel combinations ofanabolic agents, anti-catabolic agents and antioxidant agents thatmaximize beneficial, anabolic effects (healing) and minimize anypotential negative effects. In so doing, the present invention providesnovel combinations of these agents and anti-oxidant agents, for theprotection, treatment and repair of connective tissues in humans andanimals.

[0053] These compounds have a variety of beneficial effects on animaland human connective tissues, and, because they function via a varietyof mechanisms, work well in combination with each other. Although eachcompound has a number of functions, they can be roughly grouped as: (1)anabolic agents, including glucosamine, SAMe, and AA, which promotegrowth processes in the body; (2) anti-catabolic agents, such aschondroitin sulfate, pentosan sulfate, collagen type II, tetracyclinesand diacerin, which inhibit destructive or catabolic processes; and (3)antioxidants, such as SOD, and L-ergothionine which prevent tissuedamage by scavenging toxic oxygen species (free radicals). Naturally,some compounds could be placed in more than one group, by virtue oftheir overlapping functions. The present invention establishes thatcombinations of these compounds would work well. Thus, the presentinvention consists of various combinations of two or more of thefollowing agents: AA, glucosamine, chondroitin sulfate, pentosan,diacerin, S-adenosylmethionine, superoxide dismutase, L-ergothionein,collagen type II, and tetracycline-like compounds. Examples include, butare not limited to such combinations as: two anabolic agents (e.g., AAand glucosamine); an anabolic agent and an anti-catabolic agent (e.g.,glucosamine and collagen type II); an anti-catabolic and an antioxidant(e.g., tetracyclicline and superoxide dismutase); or combinations ofmore than two agents (e.g., glucosamine, SAMe and AA).

[0054] The following table shows possible combinations of pairs of thecompounds discussed above. The letter “X” marks novel combinations ofcompounds that form the novel compositions of the present invention. Theinvention also includes combinations of three or more agents of thefollowing compounds in the combinations shown on the table:

[0055] Glucosamine

[0056] Chondroitin

[0057] SAMe

[0058] Pentosan

[0059] Superoxide Dismutase (SOD)

[0060] L-Ergothionine

[0061] Collagen Type II

[0062] Diacerin

[0063] Arachadonic Acid

[0064] Tetracycline like compounds

[0065] As explained above, examples of desired combinations are markedby X. For example, the first X in the first row means a combination ofglucosamine and L-ergothionine. The compositions of the presentinvention additionally comprise any aggregation or addition of thecombinations marked by X in any given row or column. For example, thecompositions disclosed in the first row include combinations ofglucosamine plus L-ergothionine plus diacerin, or glucosamine plusdiacerin plus tetracycline-like compounds or glucosamine plusL-ergothionine plus diacerin plus AA plus tetracycline-like compounds,and so on. Examples of compositions disclosed in the column designated“Collagen Type II” would include combinations of collagen Type II plusSAMe plus pentosan, or collagen Type II plus SAMe plus pentosan plussuperoxide dismutase plus L-ergothionine, and so on. SuperoxideTetracycline Dismutase L- Collagen Arachadonic like (SOD) ErgothionineType II Diacerin Acid compounds Glucosamine X X X X Chondroitin X X X XSAMe X X X X X X Pentosan X X X X X X Superoxide X X X X X Dismutase(SOD) L- X X X X Ergothionine Collagen X X X Type II Diacerin X XArachadonic X Acid

[0066] The present inventors have investigated certain combinations ofthe above agents and have documented a novel response in severalcombinations. The effects of certain combinations of chondroitinsulfate, glucosamine, SAMe, arachidonic acid, collagen, pentosan, andsuperoxide dismutase were studied in cultures of adult bovine cartilagecells in different experiments (see example 2). Certain combinations hadan inhibitory effect (hypometabolic) in this particular study. Bothstimulatory and inhibitory novel interactions could be beneficial undervarious disease states. For example, a hypermetabolic state is part ofthe pathogenesis of some diseases. In such diseases, an inhibitory(hypometabolic) response would be beneficial to the individual. Futurestudies are planned to investigate the effects of a range ofconcentrations in the agents studied under various experimental models.Note that both increases and decreases in biosynthetic activity arenovel interactions and could be beneficial to organisms under selectedcircumstances. For example, many researchers currently believe thatosteoarthritis has a hypermetabolic component, especially in the earlystages of pathogenesis. Researchers are divided as to whether treatmentshould focus on agents that stimulate cartilage matrix production, oragents that are inhibitory and therefore make the cartilage environmentmore hypometabolic, which in turn could have a stabilizing effect on thecartilage tissue.

[0067] The compositions of the present invention may be administered viaany route, including but not limited to intramuscularly, intravenously,orally, subcutaneously, rectally, topically, transcutaneously,intranasally, and intra-articularly, sublingually, intraperitoneally.Also, any salt of any of the present compounds may be used to aid inabsorption, e.g, glucosamine HCl, glucosamine sulfate, sodiumchondroitin sulfate, etc. In addition, the composition can be given inall common dosage forms including extended release dosage forms, e.g.,pills, tablets, capsules, etc.

[0068] The dosage ranges of the compositions of the present inventionwill vary depending upon the needs of the human or animal to which thecompositions are administered. The dosage ranges for the variouscomponents of the presently claimed compositions are as follows:Compound Daily Dose Glucosamine Total dose range: 25 mg to 12g smallanimal: 25 mg-3 g. human: 100 mg-4 g large animal: 300 mg.-12 gChondroitin sulfate Total dose range: 15 mg-12 g small animal: 15 mg-2 ghuman: 75 mg-4 g large animal: 300 mg-12 g SAMe Total dose range: 10mg-8 g small animal: 10 mg-1 g human: 75 mg-3 g large animal: 400 mg-8 gPentosan Total dose range: 3 mg to 3 g small animal: 3 mg-1 g human: 50mg-2 g large animal: 100 mg-3 g Superoxide dismutase Total dose range: 3mg to 6 g (each mg containing >3000 McCord - Fridovich units) smallanimal 3 mg-2 g human: 5 mg-3 g large animal: 50 mg-6 g L-ergothioneineTotal dose range: 50 mg to 25 g small animal: 50 mg-10 g human: 50 mg-15g large animal: 100 mg-25 g Collagen Type II Total dose range: 0.1 mg to10 g small animal: 0.1 mg.-10 g human: 0.1 mg-7.5 g large animal: 1.0mg. 10 g Diacerin Total dose range: 5 mg to 5 g small animal: 5 mg-1 ghuman 20 mg-3 g large animal: 50 mg-5 g Arachadonic acid Total doserange: 10 mg to 12 g small animal: 10 mg-5 g human: 10 mg-8 g largeanimal 50 mg-12 g Tetracyclines Total dose range: 1.0 mg to 2 g smallanimal: 1.0 mg-1 g human: 2 mg-1.5 g large animal: 50 mg.-2 g

[0069] Doses are designed to cover the spectrum of body weights of smallanimals to large animals, with humans in the middle. The followingexamples are illustrative and do not in any way limit the presentinvention.

EXAMPLE 1

[0070] In our preliminary investigations, surgical instability wasinduced in the stifle joint of New Zealand white rabbits by modificationof the Hulth technique. Post-operatively, animals were exercised for 1hour daily. Experimental dietary formulas were evaluated for theircartilage stabilizing effect. The standard Harland (Teklad) rabbit diet(control); a standard diet also containing a 2% fungal oil containing40% AA by weight (Arasco); and a standard diet containing alsoarachidonic acid and glucosamine/chondroitin were investigated. At 16weeks, the medial femoral condyles of all rabbits were removed andcartilage degeneration quantitatively evaluated with a modified Mankinhistological-histochemical grading system with safranin-O stainedslides. Cartilage from all joints with surgical instability exhibitedvarying degrees of macroscopic degenerative lesions. Our preliminaryresults indicated that adding arachidonic acid toglucosamine/chondroitin sulfate has the potential to produce a novelinteraction in cartilage. This novel interaction has the potential tohave a cartilage modulating effect.

EXAMPLE 2

[0071] Procedure:

[0072] Articular cartilage was resected from human or animal jointsaseptically and placed into a large petri dish in a small amount ofDMEM/F-12 or F-12. The tissue was diced to 1-2 mm dimensions andtransferred to a small culture flask containing 20 mL DMEM or F-12+400u/mL collagenase. The flask was placed on the shaker and incubatedovernight.

[0073] The cell digest was repeatedly aspirated to increase release ofcells. The cell digest was then placed into a 50 mL sterile centrifugetube and centrifuged in the Beckman at 1000 RPM for 10 minutes. Themedium was discarded by pipette and fresh DMEM/F-12 containing 1% FCSadded. Depending on the size of the pellet, about 20-40 mL medium wasadded. Cell counts were determined by haemocytometer and the digest madeup to a concentration of 100,000 cells/0.2 mL.

[0074] GAG Synthesis:

[0075] To conduct GAG synthesis, 0.2 mL was aliquoted into each well ofa 96 well plate using an 8 channel pipetter and the cells allowed toattach for 24 hours. The media was removed and 0.3 mL of fresh 1% FCSmedia added for 2-3 days. On the day of the experiment, the media wasremoved and the experimental solutions containing 35-sulfate isotopewere added. The incubation was continued for 4 hours. Termination: atthe end of the incubation period, the labeling media was removed, thecell layer was rinsed repeatedly with cold 0.3 mL DMEM or F-12 (about5×), and the cell layer was frozen for counting.

[0076] Counting of 96 Well Plates:

[0077] The cell layer for both the synthesis experiments were heated at50 degrees after adding 100 ul 1 N NaOH for a period of 2 hours. 200 ulscintillant was added and the plates were placed in the counter. The nsulfate program was used with 1 minute counting time. The data wasexpressed as CPM/100,000 cells. Indv. Agents: Agents Evaluation CPM/ SumCombined Difference Agent 100,000 cells (CPM) (CPM) (CPM) ChSO4-L 64 AA70 134 18 −116 ChS04-H 50 AA 70 120 81 −39 Glu-H 117 AA 70 187 16 −1771% Sam 123 10 Paleos 86 209 62 −147 1% Sam 123 1 Paleos 74 197 80 −1173% Sam 42 1 Paleos 74 116 100 −16 3% Sam 42 10 Paleos 86 128 83 −45 3%Sam 42 Collagen 118 160 90 −70 3% Sam 42 AA 70 112 104 −8 AA 70 10Pentos 76 146 106 −40 Collagen 70 10 Paleos 86 156 82 −74 Collagen 11810 Pentos 76 194 65 −129 Collagen 118 10 Paleos 86 204 77 −127

[0078] In this model, at the concentrations studied, the representativecombinations had an inhibitory (hypometabolic) effect in this particularstudy. This hypometabolic effect could be beneficial under variousdisease states, indeed both stimulatory and inhibitory novelinteractions could be beneficial under various disease states. Forexample, a hypermetabolic state is part of the pathogenesis of somediseases. In such diseases, an inhibitory (hypometabolic) response wouldbe beneficial to the individual. Future studies are planned toinvestigate the effects of a range of concentrations in the agentsstudied under various experimental models. Note that both increases anddecreases in biosynthetic activity are novel interactions and could bebeneficial to organisms under selected circumstances. For example, manyresearchers currently believe that osteoarthritis has a hypermetaboliccomponent, especially in the early stages of pathogenesis. Researchersare divided as to whether treatment should focus on agents thatstimulate cartilage matrix production, or agents that are inhibitory andtherefore make the cartilage environment more hypometabolic, which inturn could have a stabilizing effect on the cartilage tissue.

EXAMPLE 3

[0079] A 4 year child has juvenile rheumatoid arthritis in which theimmune system inappropriately targets endogenous connective tissues withantibodies against native collagen type II. The resulting inflammationand degradation of cartilage causes pain and dysfunction in the synovialjoints. Present treatments include corticosteroids which non-selectivelysuppress the immune system, thus leaving the body vulnerable toinfectious disease, or methotrexate, which inhibits DNA synthesis,repair, and cellular replication, thus affecting not only the immunesystem but also intestinal mucosa, and the bone marrow. This child isgiven 2 mg of collagen type II daily, and SOD 10 mg daily. The collagendecreases the inappropriate immune attack, and the SOD inactivatesdestructive free radicals that damage cells. By preventing cellulardamage, the SOD helps maximize the normal function of joint tissuecells. This combination has no harmful side effects at therapeutic dosesand is a beneficial addition to existing therapies for rheumatoidarthritis.

EXAMPLE 4

[0080] A 6 yr old thoroughbred race horse has neutrophilic inflammationof the carpus. In this condition, trauma to the tissues of the jointinjures cells and therefore results in liberation of cytokines whichattract large numbers of neutrophils into the synovial space. Thisresponse is beneficial in cases of sepsis, but in non-septic conditionsthe neutrophils provide no useful service to the animal. Indeed, becauseneutrophils produce various degradative compounds, including superoxidemolecules, their presence in the joint contributes to a vicious cycle ofinflammation, tissue damage, and increased inflammation. Currently thiscondition is treated with nonsteroidal antiinflammatory drugs, whichsuppress prostaglandin synthesis and therefore have many side effects.This horse is given a mixture of diacerin 100 mg, pentosan 200 mg andSAMe, 1000 mg. The diacerin and pentosan both inhibit chemotaxis (theattraction of white blood cells into the affected area) and thus reducethe numbers of neutrophils in the joint. Additionally, pentosanstimulates the synthesis of synovial fluid and thus supports normalfunction of the joint. Diacerin inhibits superoxide production; sincesuperoxide production is one of the mechanisms through which neutrophilshave their harmful effects, this action of diacerin is obviouslybeneficial. SAMe supports the structure and function of cell membranes,and therefore helps repair injured joint tissue cells thus blocking theevents that start the harmful inflammation. This combination has noharmful side effects at therapeutic doses and is a great improvementover existing therapies.

[0081] One of skill in the art would understand that combinations of thecompounds taught by the present invention would act synergistically. Forexample, it is understood that glucosamine has stimulatory effects onchondrocyte metabolism which, by itself, aids in ameliorating diseasesof cartilage degradation. However, an increase in cell metabolism canalso produce an increase in free-radical production, as a naturalby-product of oxidative phosphorylation. The increase in free radicalproduction would dilute the beneficial effects of the glucosamineadministration. By combining L-ergothioneine with glucosamine, one wouldexpect an increase in metabolism and a reduction in free-radical damage,providing for a greater benefit than if compounds leading to one ofthese effects were provided. Therefore, one of skill in the art, basedon the teaching of the present invention, would understand thatcombining glucosamine with L-ergothioneine would be more beneficial thanproviding either alone. The synergy that exists between certaincompounds in the present invention also enables the use of lower dosesof each compound. Although these compounds have a quite safe, there maybe a potential for side effects. For example, large doses of glucosaminesulfate or chondroitin sulfate can cause gastrointestinal disturbancesin some individuals. In addition, these compounds are costly; for thesereasons, the ability to minimize the dose and still achieve beneficialeffects is desirable.

[0082] Many modifications may be made without departing from the basicspirit of the present invention. Accordingly, it will be appreciated bythose skilled in the art that within the scope of the appended claims,the invention may be practiced other than has been specificallydescribed herein. Hence, the attached claims are intended to cover theinvention embodied in the claims and substantial equivalents thereto.

We claim:
 1. A composition for the treatment, repair or prevention ofdamage to connective tissue comprising an aminosugar and one or morecompounds selected from the group consisting of L-ergothionine,diacerin, arachadonic acid, and tetracycline-like compounds.
 2. Acomposition for the treatment, repair or prevention of damage toconnective tissue comprising a GAG and one or more compounds selectedfrom the group consisting of L-ergothionine, diacerin, arachadonic acid,and tetracycline-like compounds.
 3. A composition for the treatment,repair or prevention of damage to connective tissue comprising SAMe andone or more compounds selected from the group consisting of SOD,L-ergothionine, collagen type II, diacerin, arachadonic acid, andtetracycline-like compounds.
 4. A composition for the treatment, repairor prevention of damage to connective tissue comprising pentosan and oneor more compounds selected from the group consisting of SOD,L-ergothionine, collagen type II, diacerin, arachadonic acid, andtetracycline-like compounds.
 5. A composition for the treatment, repairor prevention of damage to connective tissue comprising a GAG-likecompound and one or more compounds selected from the group consisting ofSOD, L-ergothionine, collagen type II, diacerin, arachadonic acid, andtetracycline-like compounds.
 6. A composition for the treatment, repairor prevention of damage to connective tissue comprising SOD and one ormore compounds selected from the group consisting of L-ergothionine,collagen type II, diacerin, arachadonic acid, and tetracycline-likecompounds.
 7. A composition for the treatment, repair or prevention ofdamage to connective tissue comprising L-ergothionine and one or morecompounds selected from the group consisting of collagen type II,diacerin, arachadonic acid, and tetracycline-like compounds.
 8. Acomposition for the treatment, repair or prevention of damage toconnective tissue comprising collagen type II and one or more compoundsselected from the group consisting of diacerin, arachadonic acid, andtetracycline-like compounds.
 9. A composition for the treatment, repairor prevention of damage to connective tissue comprising diacerin and oneor more compounds selected from the group consisting of arachadonicacid, and tetracycline-like compounds.
 10. A composition for thetreatment, repair or prevention of damage to connective tissuecomprising arachadonic acid and tetracycline-like compounds.
 11. Amethod of preventing, treating or repairing damage to connective tissuein humans and animals comprising administering the compositions of anyone of claims 1-10 to a human or animal in need thereof.